REVIEWThe roles of cell adhesion molecules on the formation of
peripheral myelin
Yasuo Takeda,1,2 Yoshinori Murakami,1,3 Hiroaki Asou1,4 and Keiichi Uyemura1,5
1Department of Physiology, School of Medicine, Keio University, 2Department of Cell Recognition, Tokyo
Metropolitan Institute of Gerontology, 3Clinical Development Department, Wyeth Lederle Japan, Ltd.
4Department of Neurobiology, Tokyo Metropolitan Institute of Gerontology, Tokyo, and 5Department of
Physiology, Saitama Medical School, Saitama, Japan(Received for publication on May 10, 2001)

Abstract. Several cell adhesion molecules of the immunoglobulin superfamily (IGSF) are expressed
differently during the development and myelination of the peripheral nervous system. To examine therelationship between the expression of IGSF molecules and Schwann cell differentiation, we establisheda useful system for myelin formation in vitro on collagen gel using primary neuron/Schwann cell cocultures from neonatal dorsal root ganglions (DRG). At 10 days in vitro (DIV), many Schwann cells
were found in the areas surrounding aggregates of DRG neurons. After 20 DIV, Schwann cells positioned next to axons and elongated their processes along the axons. Some of them started loosely
elaborating a large axon. Under electron microscopy, compact myelin was shown to be formed at 30
DIV. Thus the speed of myelination was much slower in vitro than in vivo. In co-cultures, L1 andneural cell adhesion molecule (NCAM) were detected at the premyelinating stage, L1 was precisely
expressed earlier than NCAM. Expression of myelin associated glycoprotein (MAG) was transiently
up-regulated at the early stage of myelination, and then P0 expression was ﬁnally increased as myelination proceeded. The change of expression pattern of these molecules in co-cultures was quite similar
to that observed in the development in vivo. When Schwann cell proliferation was blocked by low
serum culture condition, L1 and NCAM expressions were up-regulated. In contrast, the presence ofcholera toxin in low serum media markedly increased expressions of P0 and MAG, but decreased thelevels of both L1 and NCAM. These results suggest that both L1 and NCAM play roles in the contact
and/or recognition between axons and Schwann cells at an early stage of myelination. On the other
hand, MAG and P0 are important for axon ensheathment and myelin compaction. (Keio J Med 50 (4):240–248, December 2001)
Key words: Schwann cells, myelination, PNS, cell adhesion molecules

Introduction
One of the important developmental events in the
nervous system is the differentiation of glial cells toform myelin. Myelin is a highly specialized multilamellar
membrane that results from the elaboration of plasma
membranes of Schwann cells in the peripheral nervous
system (PNS) or oligodendrocytes in the central nervous system. To form and maintain a precise myelin
structure is very important in its function as an insulator
to increase the velocity of nerve conduction in axons.
Disruption of this structure is well known to cause se-vere neurological diseases such as multiple sclerosis andperipheral neuropathies.1,2
In the case of the PNS, the task of the Schwann cell,which originates from a neural crest cell, is to form peripheral myelin.3 The differentiation of Schwann cells
causes a dramatic morphological change in the cells.4
The process of myelination is largely regulated byprotein-protein interactions between the axon and the
Schwann cell. It has been suggested that further differentiation of Schwann cells is precisely regulated by achain expression of myelin speciﬁc proteins. Some ofthese proteins and gangliosides have been identiﬁed as

Reprint request to: Dr. Yasuo Takeda, Department of Cell Recognition, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173-0015, Japan, e-mail: yachan@center.tmig.or.jp240

Keio J Med 2001; 50 (4): 240–248

markers for Schwann cell differentiation. S-100 protein
is expressed in immature Schwann cells in the proliferative stage. As Schwann cell differentiation proceeds,
O4, glial ﬁbrillary acidic protein and galactocerebrosideare expressed successively as markers.5
Many reports have demonstrated that cell adhesion
molecules are spatio-temporally expressed in the nervous tissues during development and play critically important roles for the proper cell-cell interactions.6,7
Several characteristic proteins of cell adhesion molecules that belong to the immunoglobulin superfamily(IGSF) have been identiﬁed as regulating peripheral
myelin formation. P0, having a single V-like external
domain, is afﬁliated with the IGSF.8 It is exclusively
expressed in myelin-forming Schwann cells and is localized in the compacted regions of mature myelin.9 It
has been proposed that P0 exhibits strong homophilicbinding and plays a role in the compaction of the multilamellar membrane.10,11 In contrast, L1 and neuralcell adhesion molecules (NCAM) are expressed in bothneurons and Schwann cells, suggesting that they playa role in cell-cell contact as recognition molecules.12
However, it has not been well understood how the expression of these molecules is linked to myelination.
To understand the mechanism of myelination, an
important ﬁrst step is to elucidate the precise relationship between the expression pattern of cell adhesion
molecules and the morphological and functional differentiation of Schwann cells to form compact myelin.
For this purpose, we established a useful system for myelination on collagen gel by using dorsal root ganglion(DRG) neuron/Schwann cell primary co-cultures. We
have carried out high resolution electron microscopy,
immunocytochemistry and an enzyme-linked immunosorbent assay (ELISA) to examine changes in the gene
expression and morphology of Schwann cells.
Materials and Methods
Materials
Collagenase/dispase and aprotinin were purchasedfrom Boehringer-Mannheim. Dulbecco’s modiﬁed
Eagle’s medium (DMEM), 5-ﬂuoro-5 0 -deoxyuridine,
cytosine arabinoside, cholera toxin, XTT; 2,3-bis (2methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carboxyl]-2H-tetrazolium hydroxide, Menadine; methyl1,4-naphthoquinone sodium bisulﬁte, phenylmethylsulfonyl ﬂuoride, pepstatin, and bovine serum albumin(BSA) were purchased from Sigma. Trypsin and polyL-lysin (PLL) were purchased from Difco laboratories,
Dulbecco’s phosphate-buffer saline (PBS) was purchased from Nissui, 7S-Nerve growth factor (7S-NGF)
was purchased from Chemicon, L-ascorbic acid 2phosphate were purchased from Wako Pure Chemicals,

241

Type I collagen (Type I-A) was purchased from Nitta
gelatin Co., fetal calf serum and horse serum were purchased from Flow laboratories, and goat serum was purchased from Biomakor. Anti-myelin associated glycoprotein (MAG) antiserum and anti-NCAM antiserum
were a generous gift from Dr. Sato at Niigata University and Dr. Ono at Shimane Medical College, respectively. Antibodies against L1 and P0 were generated in
our laboratory. Other antibodies were purchased fromeither E-Y Laboratories or Biomakor.
Schwann cell culture
Neonatal Wistar rat sciatic nerves were dissected andtreated with 1% collagenase/dispase in DMEM for 1
hour at 37  C. The reaction was stopped with a 10-foldvolume of normal culture media containing 0.5% glucose, 0.045% sodium bicarbonate, 0.002% kanamycin
and 10% fetal calf serum (FCS), and then cells weredispersed by pipetting. After centrifugation at 120  g
for 5 min, cells were resuspended with normal culture
media. This process was repeated twice to clean the cellsuspension. Finally cells were seeded on PLL precoated 35 mm diameter plastic dishes and cultured for
24 hours under normal conditions. On the next day,cultures were treated with 105 M cytosine alabinosideand maintained for a further 1 week to eliminate ﬁbroblast cells. Schwann cells were then allowed to proliferate in response to 20 ng/ml cholera toxin.13 For thecell growth assay or ELISA, Schwann cells were incubated with 2% FCS-containing media for 2 weeks without cholera toxin.
Schwann cell growth assay
To measure cell growth activity, 1:2  10 4 Schwann
cells were seeded in each well of a 96-well plate. Cells
were subsequently allowed to grow for 7 days underfour different conditions. The medium was refreshedonce at 4 days after the start of incubation. At the endof incubation, cells were treated and lysed with 0.025
mM XTT and 0.2 mM Menadine. The absorption of agenerated formazan in each well was measured at 450
nm using a micro-titer plate reader, and the opticaldensity was evaluated as a marker of the density of
Schwann cells.14
DRG neuron and Schwann cell co-cultures: myelin formation in vitro
DRGs of Wistar rat embryos at 19 days of gestation
were isolated by dissociation with collagenase/dispase.
The dispersed cells were ﬁnally resuspended with anormal culture medium at a density of 1  10 6 cells/ml
and plated onto collagen gel prepared in a 35 mm dish

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according to the manufacturer’s method. On the nextday, cultures were treated with a one 3-day pulse of
1 mM ﬂuoro-deoxyuridine to eliminate non-neuronalcells and then maintained in a normal culture medium
containing 100 ng/ml 7S-NGF and 50 mg/ml L-ascorbic
acid 2-phosphate for as long as 40 days.
DRG neuron and Schwann cell co-cultures: immunocytochemistryCo-cultures grown on glass coverslips coated with
PLL were immunostained for L1, MAG, P0 and laminin using standard protocols. Brieﬂy, the cells wererinsed in PBS several times and ﬁxed by incubation
with PLP [0.0375 M phosphate buffer (pH 6.2) containing 0.075 M lysine, 2% paraformaldehyde and 0.01 M
sodium metaperiodate] for 10 min at room temperature.
Non-speciﬁc binding was blocked by pre-incubation with
PBS containing 10% BSA and 1% horse serum. All
immunostaining was carried out with a 1 : 500 dilution ofpolyclonal antibodies. A 1 : 1,000 dilution of ﬂuoresceinconjugated anti-rabbit goat serum was used as the second antibody. Co-cultures were mounted and wereviewed on a Zeiss universal microscope equipped withﬂuorescence optics.
Electron microscopy
The neuron/Schwann cell co-cultures of DRG grownon collagen gel were ﬁxed in buffered 2% glutaraldehyde followed by 2% osmium oxide, dehydrated
in ethanol and embedded in Epon 812. Areas to be
examined were selected and thin sectioned for electron
microscopy. Thin sections were stained with uranyl
acetate and lead citrate and viewed under a Hitachi H700 electron microscope.
For the electron microscopic immunocytochemical
examination, the co-cultures were ﬁxed in PLP, treatedwith a blocking solution, incubated with a primarypolyclonal antibody against to P0 overnight at 4  C,
followed by incubation with peroxidase-conjugated secondary antibody for 1 hour at room temperature. Afterseveral rinses, co-cultures were further ﬁxed in 2.5%
glutaraldehyde for 5 min, and then stained with 3,3 0 diaminobenzidine. The ultra thin sections were made by
the identical procedure described above.
ELISA(1) Sampling: DRG neuron/Schwann cell co-cultureson collagen gel and Schwann cells grown in T-75 ﬂasks
were washed in PBS several times, harvested with a cell
scraper and lysed in 1–3 ml lysis buffer (20 mM Tris
phosphate buffer pH 8.3, 150 mM sodium chloride,
1 mM EDTA, 0.01 mM pepstatin, 0.2 unit/ml aprotinin,

Takeda Y, et al: Myelination and cell adhesion molecules

1 mM phenylmethylsulfonyl ﬂuoride) by sonication.
After centrifugation at 100,000  g for 1 hour at 4  C,
the supernatant was used for ELISA. To examine thedevelopmental change of the expression level of celladhesion molecules in vivo, the ELISA sample was prepared from Wistar rat sciatic nerve tissue at embryonicday 19 (E19), postnatal days 0 (P0), 4 (P4), 7 (P7), and 10(P10). Dissected sciatic nerves were homogenized witha polytron mixer and centrifuged supernatants wereused for the ELISA.
(2) Assay: Each ELISA sample was diluted 3–10
fold with a borate buffer pH 7.6 (50 mM boric acid, 2
mM sodium tetraborate) and 100 ml of each was coatedonto each well of 96-well plates. After 1 day incubation
at 4  C, the coated plates were washed with PBS. The
wells were then blocked for 1 hour at room temperature with a blocking buffer (described above). After
blocking, 100 ml each of primary antibodies was added
to the wells and incubated at room temperature for 1
hour. The unbound antibodies were aspirated, followedby three washes with PBS containing 0.05% Tween20.
The 100 ml of 1,000-fold diluted secondary antibody(either horseradish peroxidase-conjugated anti-rabbit
IgG or horseradish peroxidase-conjugated anti mouse
IgG) was added to each well and incubated at room
temperature for 1 hour. At the end of incubation, thewells were washed, followed by incubation with 100 mlof phosphate-citrate buffer pH 5.0 (24.3 mM citric acid,
51.4 mM disodium hydrogenphosphate) plus 0.2% Ophenylene diamine and 0.02% hydrogen peroxide to
develop color for 30 min at room temperature. At theend of incubation 100 ml of 4.5 M sulfuric acid wasadded to each well to stop the color reaction. The optical density of each well was determined at 450 nm using
a spectrophotometer.
Results
Expression of cell adhesion molecules in Schwann cells
under different culture condition
After incubation of neonatal Schwann cells with 2%
FCS-containing basal media for 2 weeks, the cells were
cultured under 4 different culture conditions for 1 week.
When the cells were exposed to 10% FCS-containing
media, the density of the cells increased up to 3-foldthat of the initial level (Fig. 1). In the presence of cholera toxin in 10% FCS-containing media, the cell density
increased additionally up to 6-fold. In contrast, in 2%
FCS-containing media the cell density was not affectedby the addition of the cholera toxin.
By ELISA analysis, expression levels of L1, NCAM,
MAG, P0 and laminin were examined on each lysateof Schwann cells cultured under 4 different conditions.
In the cultures exposed to 10% FCS-containing media

Keio J Med 2001; 50 (4): 240–248

Fig. 1 Expression of cell adhesion molecules in Schwann cells under
different culture condition. Schwann cells were cultured for 1 week
under 4 different conditions, (1) 10% FCS, 10% FCS-containing
DMEM, (2) 10% þCTx, 10% FCS-containing DMEM plus 100 ng/ml
cholera toxin, (3) 2% FCS, 2% FCS-containing DMEM, (4) 2%
þCTx, 2% FCS-containing DMEM plus 100 ng/ml cholera toxin.
After a week incubation, samples were lysed. The respective content
of L1, NCAM, MAG, P0 and laminin was measured by ELISA anddescribed as a relative expression level to the ‘Initial’ that is cells before the week incubation. Three hours before the end of culture, XTT
and Menadine were added and the cell density of each sample wasmeasured for the optical density of formazan. Each value was calculated as an average for 3 or 4 independent experiments. Bars describe
standard deviation (SD) values.

with or without cholera toxin, the expression levels of
all ﬁve molecules were not signiﬁcantly changed. In
contrast, the levels of L1 and NCAM were increased byabout 4–5 fold when the cells were exposed to the 2%
serum condition. Additional exposure to cholera toxin
in 2% serum medium markedly increased the expression of both MAG and P0 up to 20-fold of the initiallevel, while the levels of L1, NCAM and laminin were
not signiﬁcantly affected.
Immunocytochemical and morphological observationsof myelin formation in vitro
The time course of myelin formation in vitro wasstudied in the co-cultures of DRG neuron and Schwann
cell. At 4 days culture in vitro (DIV), spindle shaped
Schwann cells were markedly immunostained with antiS-100 antibody (Fig. 2A). The DRG neuron was recognized as a large round cell body in the co-culturesby phase contrast microscopy (Fig. 2B). This large
rounded cell body of the DRG neuron was weakly
immunostained with anti-S-100 antibody, a marker for
the Schwann cells, suggesting its non-speciﬁc staining.
In contrast, no immunoreactivity was detected in ﬂatshaped ﬁbroblast cells. The expression of L1 was ob-

243

Fig. 2 Expression of S-100 and L1 in DRG neuron/Schwann cell
co-cultures at 4 DIV. The cells co-cultured on PLL-coated glass
coverslips were immunostained for either (A) S-100 or (C) L1, (A)Immuno-microscopic observation of co-cultures at 4 DIV for S-100
like immunostaining, (B) Phase contrast observation of an identicalarea of A. A short arrow indicates a Schwann cell immunostainedwith anti-S-100 antibody. Arrow head in panels A and B shows the
DRG neuron weakly immunopositive to anti-S-100 antibody, (C)Immuno-microscopic observation of co-culture at 4 DIV for L1 likeimmunostaining, (D) Phase contrast observation of an identical areaof C. The long arrow or arrow head indicates respectively a Schwann
cell or DRG neuron immunoreacted with anti-L1 antiserum in panels
C and D. It was also shown that ﬁbroblast cells were immuno-negative
for antibodies against either S-100 or L1. Bars describe a 200 mm
length.

served in both DRG neurons and Schwann cells, but
not in ﬁbroblast cells at the stage of 4 DIV (Fig. 2C). At
10 DIV, many Schwann cells surrounding aggregates of
DRG neurons were observed (data not shown).
At 20 DIV, DRG neuron neurites were grown andwere attached side by side with Schwann cells (Fig. 3).
Most of the Schwann cells at this stage contained elongated nuclei and extended long processes along theaxons. Laminin was localized in the extents of the
Schwann cell cytoplasm at this stage (Fig. 3A). On
the other hand, MAG expression appeared to be upregulated in some but not in all of the Schwann cellprocesses (Fig. 3C). Under high resolution electron
microscopy, Schwann cells were seen to develop many
plasmalemmal domains that processed and/or engulfed
large axons (Fig. 4A). In addition, the formation of basal
lamina surrounding the Schwann cell/axon complex was
also observed. Some of the differentiated Schwann cells
formed a mesaxon that spiraled up to 2 turns around anaxon at this stage of 20 DIV (Fig. 4B).
Compact myelin was revealed in co-cultures at 30
DIV (Fig. 5). A large diameter axon spiraled with 11
layers of compacted lamellae is shown in ﬁgure 5A. A
bunch of small rounded microtubules was found inside
the axon. Many cases of segregated large axons were244

Takeda Y, et al: Myelination and cell adhesion molecules

Fig. 3 Expression of laminin and MAG in DRG neuron/Schwann
cell co-cultures at 20 DIV. The cells co-cultured on PLL-coated glass
coverslips were immunostained for either (A) laminin or (C) MAG,(A) Immuno-microscopic observation of co-cultures for laminin at 20
DIV, (B) Phase contrast observation of an identical area of A. Many
immuno-positive Schwann cells are shown. A short arrow indicates
the weakly immunoreacted ﬁbroblast cell, (C) Immuno-microscopic
observation of co-culture for MAG at 20 DIV, (D) Phase contrast
observation of an identical area of C. A long arrow indicates the
Schwann cell body and its process that were immunoreacted with antiMAG antiserum. Bars ¼ 200 mm.

associated 1 : 1 with Schwann cells and were wrappedwith 10–30 layers of compact myelin sheath. Under preembedding immunoelectron microscopy, P0 was shown
to be localized in the compacted lamellae (Fig. 5B).
Temporal expression of cell adhesion molecules during
peripheral myelin formation: in vivo and in vitro
To examine the expression patterns of several celladhesion molecules in DRG neuron/Schwann cell cocultures, their contents at different stages in vitro were
determined by ELISA. Patterns of their expression
were additionally compared to those in vivo (Fig. 6).
As for L1, the highest content of L1 of 0.9 ng/mg
protein was detected in 10 DIV at the time when DRG
neuron/Schwann cell interaction started. L1 content
was subsequently signiﬁcantly decreased to one-half the
maximum level at 20 DIV and was maintained at thislow level till 40 DIV. The L1 content of sciatic nerve at
E19 in vivo was as high as 0.7 ng/mg protein, following
which its expression was developmentally decreased to
the level of one-quarter of the maximum at P7 andmaintained at this low level till P10 (Fig. 6A).
The NCAM content in co-cultures was 0.77 ng/mg
protein at 10 DIV and transiently but not signiﬁcantly
increased in 20 DIV at the time of myelination onset,
and then decreased to less than one-half the maximum
level at 40 DIV. In vivo, the peak (1.0 ng/mg protein)

Fig. 4 Ultrastructural observation of a morphologically changed
Schwann cell in co-cultures at 20 DIV. Co-cultures maintained for 20
DIV on the collagen gels were observed by electron microscopy.
Transverse sections through an area of a co-culture that contains few
Schwann cells interacting with axons are illustrated, (A) Many casesof Schwann cell membranous protrusions in interspace of the segregated axons are seen. Besides the formation of basal lamina surrounding Schwann cell was evident at this stage. An asterisk indicatesa formed basal lamina surrounding Schwann cell/axons complex, (B)Several very thin membranous with no compacted myelin are
revealed around the axons. The arrow indicates a mesaxon thatspiraled up to 2 turns around an axon. Bars ¼ 1 mm (A), 5 mm (B).
Abbreviations: SC, Schwann cell; Ax, axon of DRG neuron.

was detected at P4. It was about twice as much as thelevel at E19. The level of NCAM subsequently quickly
reduced at P10 to an eighth of the peak (Fig. 6B).
The MAG content was 0.4 ng/mg protein at 10 DIV
and was transiently up-regulated to 1.5-fold at 30 DIV.
In in vivo development, the expression level of MAG at
the embryonic stage was low. It quickly increased after
birth, peaked at P7 up to a 6-fold level and slightly decreased at P10 (Fig. 6C).
On the other hand, expression of P0 dramaticallyincreased as co-cultures proceeded in vitro. Upregulation of P0 expression coincided with morphological dif-

Keio J Med 2001; 50 (4): 240–248

Fig. 5 Ultrastructural observation of compact myelin in co-culturesat 30 DIV. Co-cultures maintained for 30 DIV on the collagen gelswere observed by electron microscopy, (A) Transverse section
through an area of a co-culture that contains an 11-layered compact
myelin sheath is shown. Most cases seen in cross-sectional proﬁle ofmyelin sheaths contained compacted lamellae that are spiraled up
between 10–30 layers, (B) P0 localization in compacted myelin in
vitro shown by immunoelectron microscopic observation. Arrow
indicates P0-like immunoreactivity seen as a black dot which is localized in the compacted lamellae. Bars ¼ 1 mm length.

ferentiation of Schwann cells. In the case of sciatic nerve
development in vivo, P0 was only detectable before P4
and was markedly increased in 50-fold at P7 and P10,
suggesting a close relationship with compact myelin formation (Fig. 6D).
Discussion
The molecular mechanism of myelin formation has
not been well understood, while expressions of celladhesion molecules are regulated spatio-temporally inboth axons and myelin-forming cells during myelination.6,7 In this study, we established a co-cultures system of DRG neurons and Schwann cells on collagen gel
and examined the developmental change of adhesion

245

molecule expression during Schwann cell differentiationin vitro. In addition, the expression pattern in vitro wascompared to that in vivo.
When Schwann cell proliferation was inhibited in alow serum medium in vitro, L1 and NCAM expressions
were up-regulated. In the presence of cholera toxin,
however, their expression levels were conversely downregulated and the expression of both MAG and P0
markedly increased. These results suggested that theexpression of L1 and NCAM was correlated with the
inhibition of Schwann cell proliferation. On the other
hand, an increase of intracellular c-AMP at growth arrest potently induced P0 and MAG expression.
Most Schwann cells proliferate actively at the earlystage of co-cultures with DRG neurons and at the latestage only the cells surrounding the distal end of theextended axon are still in the proliferative stage.15 In
the case of PNS development in vivo, it has been suggested that L1 and NCAM expressions are up-regulated,
as proliferation of the Schwann cells is reduced. On theother hand, the effect of intracellular c-AMP content
on P0 expression has been complicated. Treatment of
Schwann cells from neonatal rat sciatic nerve with folskolin or glial growth factor, both of which activate
adenylate cyclase, increases markedly the expression of
P0 mRNA and protein.16,17 On the other hand, no effect of folskolin on P0 expression has been reported.18
In the latter case, the prepared Schwann cells derived
from either neonatal rat sciatic nerves or Waller degenerated nerves in the adult rat were still proliferative. As
shown in this study, cholera toxin induced up-regulationof P0 expression when Schwann cells were grown in
2% FCS-containing medium, but not in 10% FCS
which usually maintains the proliferation of Schwann
cells. Taken together, P0 expression depends on both
Schwann cell growth arrest and regulation of intracellular c-AMP content. MAG expression seems to be
likely in the case of P0. The expression level of laminin
was not affected by Schwann cell growth arrest.
In order to examine myelin formation in vitro, cocultures were carried out on collagen gels instead of
PLL-coated plastic dishes. Bunge et al. have reported
that an extended axon did not attach well to the surface
of a PLL-coated dish so that Schwann cells could notadhere to the axon under this condition.19 When thecollagen gel was used as a culture substrate, Schwann
cells attached well to the extended axon. Additionally L-ascorbic acid, which is a co-enzyme to prolylhydroxylase for collagen synthesis, is known to be necessary for the formation of myelin in vitro.20 To develop
an extracellular matrix structure for myelin formationin vitro, the cells have to produce collagen by themselves to interact with laminin. In this study, the Lascorbic acid 2-biphosphate was used to avoid oxidation
instead of ascorbic acid.

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Takeda Y, et al: Myelination and cell adhesion molecules

Fig. 6 Temporal expression patterns of several cell adhesion molecules in vivo and in vitro during peripheral myelin formation. Each panel
shows a developmental change of either (A) L1, (B) NCAM, (C) MAG or (D) P0 expression in co-cultures in vitro compared to that in sciatic
nerve in vivo. The content of each molecule in samples prepared from either co-cultures or sciatic nerves at the indicated days was measured by
ELISA and was calculated as a ng/mg protein content in each preparation. Data are based on four independent experiments. Statistics within
ﬁgures (mean G standard deviation) were compared by Student t-test and signiﬁcantly different statistics are indicated by asterisks (*, p < 0:05;**, p < 0:01).

Under electron microscopy, we observed two membranous spirals around an axon at 20 DIV and a compacted myelin sheath with more than 10 lamellae at 30
DIV. At the onset of myelination of the sciatic nerve invivo, Schwann cells segregate large axons away fromsmaller axons, which will remain unmyelinated, and invest the large axons with a single layer of membrane.
The Schwann cells initiate a spiral wrapping around asingle large axon and wind it up with uncompacted
lamellae of 3 or 4 layers at P3. At P7, many compacted
lamellae were observed surrounding the axons, which
consisted of 17 to 28 layers of membrane. This indicatesthat each myelination stage at 20 DIV and 30 DIV in
co-cultures corresponds to the respective myelination
stage at P4 and P7 in vivo.
The expression pattern of cell adhesion moleculeschanged remarkably at either 20 DIV in vitro or P4 in

vivo. At this timing, onsets of myelination are observed.
Both L1 and NCAM are highly expressed before theonset, and are subsequently quickly down-regulated. In
contrast, the expression of both P0 and MAG are upregulated as myelin formation proceeds. It seems thatthe onset of myelination is very important to regulate
the expression level of cell adhesion molecules. Sequential expression peaks of each L1, NCAM, MAG and P0
are observed during myelination.
L1 is known to be expressed in both neurons andglial cells. A full length of L1 is expressed in neurons,
while a short form that lacks exons 2 and 27 is only
expressed in non-neuronal cells.21,22 Exon 27 encodesfor 4 amino acids (RSLE) the sequence of which is important for cell migration and L1 recycling in the growthcone.22,23 Myelinating Schwann cell membranes thatform the initial one and a half spiral wraps around anKeio J Med 2001; 50 (4): 240–248

axon are also L1-positive.24 As myelination proceeds,
L1 is no longer detected in Schwann cell membranes.
L1 antibodies inhibited the myelination of DRG axons
maintained in vitro,25,26 suggesting that L1 is necessary
for axonal ensheathment and initial spiral wrapping by
the myelin membranes. NCAM is also expressed inboth DRG neurons and Schwann cells and is suggested
to enhance an L1-L1 homophilic adhesion.27 As myelination proceeds, MAG may function to form uncompacted lamella membranes by possibly either homo- or
heterophilic interaction, and ﬁnally P0 works for compaction to form a multilamellar membrane structure via
P0-P0 adhesion.
Based on previous studies in mice deﬁcient for thecorresponding molecules, only P0 plays pivotal roles
during peripheral myelin formation. A disruption of theP0 gene in mice leads to a severe dysmyelinating neuropathy with predominantly uncompacted myelin.28 P0
is the gene responsible for peripheral neuropathies inhumans, such as Dejerine-Sottas disease and CharcotMarie-Tooth disease type 1B.2,29,30 In contrast to a severe hypomyelination in P0 knockout mice, L1, NCAM
and MAG-deﬁcient mice develop normal peripheral
myelin.31–33 However, MAG turned out to be important for the maintenance of the myelin structure.33
Analyses of P0/NCAM- and P0/MAG-deﬁcient double
mutants mice revealed that an absence of NCAM
resulted in a transient retardation of Schwann cell spiraling, whereas absence of MAG impaired Schwann cellspiraling for a more extended time period, comparedto P0-deﬁcient mutant mice.34 On the other hand, twolines of L1 null mice showed normal myelination, indicating that L1 is not essential for initial axon ensheathment or myelination.31,35 Later on, L1-deﬁcient mice
showed loss of unmyelinated sensory axons, resulting in
reduced sensory function, while sympathetic unmyelinated axons appeared normal.36 Through nerve transplant studies, adhesion between Schwann cells andaxons is disrupted in the case of L1-positive Schwann
cells transplanted into L1-deﬁcient nerves, as well as in
the case of absence of L1 in both axons and Schwann
cells. However, Schwann cell-axon adhesion is maintained when L1-deﬁcient Schwann cells are transplanted
into L1-positive nerves.36 The data suggest that heterophilic axonal-L1 interactions mediate adhesion between unmyelinated sensory axons and Schwann cells.
According to this result, there may exist unknown molecule(s) for the initial event of axon ensheathment.
In conclusion, the cell adhesion molecules L1,
NCAM, MAG and P0, which all belong to the immunoglobulin superfamily, seem to have a chain expression in the mediation of peripheral myelin formation.
L1 probably plays a role co-operatively with NCAM
for the recognition and adhesion between axons and
Schwann cells at the premyelinating stage. On the other247

hand, MAG and P0 are important for axon ensheathment and myelin compaction, respectively.
Acknowledgements: We are greatly indebted to Ms. Junko Takeda
for her technical help and indispensable advice. We also thank Mr.
Yoshihiro Fujita for his photography. This work was supported in partby research grants from the Ministry of Education, Science, Sports
and Culture of Japan.

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